宁夏枸杞PLA基因家族cDNA片段的克隆及其生物信息学分析

doi:10.7525/j.issn.1673-5102.2014.06.017

摘要/Abstract

摘要： 苯丙氨酸解氨酶（PAL，EC 4.3.1.5）是一种主要的调控酶，在植物体内通过催化L-苯丙氨酸生成肉桂酸来联系初级和次级代谢。本文以宁夏枸杞为材料，利用序列同源原理设计简并引物和RT-PCR技术得到4条PAL基因片段，并对其进行生物信息学分析。结果表明，该家族的cDNA片段长为1 162~1 183 bp，推导编码554~561个氨基酸，蛋白分子量为60.681~61.250 kD，等电点为7.02~7.71。枸杞PAL基因家族彼此间的氨基酸序列变异度为0.005%~0.336%。氨基酸序列和结构分析显示PAL基因家族有一个保守区域，即屏蔽结构域，以及一个活性位点——苯丙氨酸/组氨酸解氨酶位点。LyPAL1~3蛋白很可能定位在线粒体，而LyPAL4蛋白很可能定位在细胞质。二级结构和三级结构进行预测，发现LyPAL1蛋白中无规则卷曲179个，α螺旋和β折叠分别306和30个，延伸链46个。系统进化分析表明，枸杞LyPAL1~3基因与辣椒的PAL基因具有很高的同源性，而枸杞LyPAL4基因与碧冬茄属的三种植物（Petunia axillaris，Petunia x hybrid，P.exserta）具有很高的同源性。这4个基因已在GenBank上注册，基因序列登录号为KC759153~KC759156。4个LyPAL基因的克隆为进一步研究宁夏枸杞类黄酮等次级代谢物的合成分子机制研究奠定了基础。

关键词: 枸杞, PLA基因, 克隆, 生物信息学分析

Abstract: Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is a key regulatory enzyme that link primary and secondary metabolism in plants by catalyzing the conversion of L-phenylalanine to cinnamic acid. Four partial sequences of PAL in Lycium barbarum L. were successfully cloned by RT-PCR using a sequence homology strategy, and the bioinformation analysis were carried out. The cDNA fragment of PAL gene family was 1 662-1 683 bp in length, which encodes proteins of 554-561 amino acids with the predicted molecular mass of 60.681-61.250 kD. The amino acid sequence variation between the PAL gene families is 0.005%-0.336%. The estimated isoelectric point (pI) of the putative protein is from 7.02 to 7.71. According to the amino acid sequence and structural analysis, it showed that this protein family contained one conserved domain, namely the shielding domain, and one active site which is phenylalanine/histidine ammonia-lyases. Subcellular localizations of LyPAL1-3 proteins were likely in the mitochondrion and LyPAL4 in the cytoplasm. The secondary structures and tertiary structures of LyPAL1 were abundant in alpha helixs (306) and random coils (179), while were less in beta turns (30) and extended strains (46). Phylogenetic analysis showed that the genes of LyPAL1-3 in L.barbarum L. were closely related to PAL in Capsicum annuum, while LyPAL4 was closely related to PAL in Petunia Juss. (Petunia axillaris, Petunia x hybrid, P.exserta). These four sequences have been registered in GenBank with the accession numbers KC75915-KC759156. Four LyPAL were cloned by molecular biology technology, which would contribute to the molecular mechanisms of the formation of flavonoids and other secondary metabolites in L.barbarum L..

Key words: Lycium barbarum L., PLA gene, cloning, bioinformation analysis

中图分类号:

陈华峰1；陈阿丽2；尚洁3*；张凤娇4. 宁夏枸杞PLA基因家族cDNA片段的克隆及其生物信息学分析[J]. 植物研究, 2014, 34(6): 816-823.

CHEN Hua-Feng1；CHEN A-Li2；SHANG Jie3*；ZHANG Feng-Jiao4. Cloning and Bioinformation Analysis of PLA Gene Family in Lycium barbarum L.[J]. Bulletin of Botanical Research, 2014, 34(6): 816-823.

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